Preparation method and application of nucleic acid aptamer modified nano-enzyme ion imprinting polymer

Abstract

The application relates to a preparation method and application of a nucleic acid aptamer modified nanoenzyme ion imprinting polymer and belongs to the technical field of medical detection. 2+ The nucleic acid aptamer, a sodium salt, are co-incubated to obtain a non-imprinted polymer; step 3: the nucleic acid aptamer modified nanoenzyme obtained in step 2 is pre-assembled with Pb 2+ A functional monomer is then added and polymerization is initiated, and the precipitated particles are obtained through centrifugal separation. Step 4: the centrifugal product is washed with an acid solution to remove Pb 2+ , and the nucleic acid aptamer modified nanoenzyme ion imprinting polymer is obtained. The imprinting layer formed by ion imprinting on the nanoenzyme interface stabilizes the binding conformation of the nucleic acid aptamer and the template ion, overcomes the interference of environmental factors on the nucleic acid aptamer and the non-specific adsorption behavior, and improves the recognition selectivity of the nucleic acid aptamer modified nanoenzyme.

Description

Technical Field

[0001] This invention relates to a method for preparing and applying a nucleic acid aptamer-modified nanoenzyme ion-imprinted polymer, belonging to the field of biosensing technology. Background Technology

[0002] Nanozymes have attracted widespread attention due to their excellent enzyme-like activity, simple preparation, high environmental stability, and low cost. Furthermore, as signal reporter units, nanozymes can efficiently catalyze the production of color or fluorescence signals from substrates. However, the lack of specific recognition units on the surface of nanozymes limits their sensing applications. Aptamers are nucleic acid fragments with specific recognition capabilities; modifying them onto the surface of nanozymes enables them to specifically recognize targets. However, the inherent flexible structure of nucleic acid aptamers, their target-binding conformation, and adaptive folding behavior are easily affected by environmental factors such as pH and ionic strength, leading to a significant reduction in their recognition and binding ability in complex matrix environments, or even their inability to bind. In addition, the non-specific adsorption behavior of immobilized nucleic acid aptamers at interfaces also affects their recognition and the catalytic properties of nanozymes.

[0003] Therefore, there is an urgent need in this field for a nucleic acid aptamer-modified nanoenzyme ion-imprinted polymer that has good selective recognition ability and whose recognition of the aptamers modified on its interface is not easily affected by environmental and non-specific adsorption. Summary of the Invention

[0004] The problem to be solved by the present invention is that the selective recognition ability of nanozymes in the prior art is poor, and the recognition performance of the aptamers modified on their interface is easily affected by the environment and non-specific adsorption.

[0005] To address the aforementioned problems, the present invention provides a method for preparing and applying a nucleic acid aptamer-modified nanozyme ion-imprinted polymer.

[0006] In a first aspect, the present invention provides a method for preparing a nucleic acid aptamer-modified nanozyme ion-imprinted polymer, comprising the following steps:

[0007] Step 1: Preparation of gold nanoparticles;

[0008] Step 2: Combine the prepared gold nanoparticles with Pb 2+ Nucleic acid aptamers are co-incubated, sodium salt is added, and incubation is repeated. Excess nucleic acid aptamers are removed by centrifugation to obtain nucleic acid aptamer-modified nanozymes (Au-T30695).

[0009] Step 3: Combine the aptamer-modified nanozyme obtained in Step 2 with Pb 2+ Pre-assembled, then functional monomers are added and polymerization is initiated, and finally centrifuged to obtain precipitated particles.

[0010] Step 4: Wash the centrifuged product with an acidic solution to remove Pb. 2+ Until no Pb is found in the washing solution. 2+ Upon detection, the nucleic acid aptamer-modified nanozyme ion-imprinted polymer Pb-IIP is obtained.

[0011] Preferably, step 2 satisfies one or more of the following conditions:

[0012] Condition 1 is gold nanoparticles and Pb 2+ The molar ratio of nucleic acid aptamers is 1:30 to 1:500;

[0013] Condition 2 is that the co-incubation time is 20h to 24h;

[0014] Condition 3 is that the concentration of the sodium salt is 0.1M;

[0015] Condition 4 is that the additions are made every half hour, for a total of four times;

[0016] Condition 5 is that the incubation time is 40-48 hours.

[0017] Preferably, in step 3, the concentration of the functional monomer is 0.01–1 mg / mL. -1 The polymerization time is 30 min to 360 min, and the functional monomer is further preferably dopamine.

[0018] A second aspect of the present invention provides a method for using the above-mentioned nucleic acid aptamer-modified nanozyme ion-imprinted polymer on Pb 2+ The application in the detection includes the following steps:

[0019] Nucleic acid aptamer-modified nanozyme ion-imprinted polymer Pb-IIP with different concentrations of Pb 2+ The solutions were mixed, followed by the addition of Amplex UltraRed fluorescent substrate and H2O2 for co-incubation. Fluorescence intensity at 584 nm was recorded under 540 nm excitation to establish a system for different concentrations of Pb. 2+ The linear relationship between the fluorescence intensity of the solution and the change in fluorescence intensity.

[0020] Preferably, the co-incubation temperature is 25–37°C and the time is 2 hours.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The imprinted layer formed by ion imprinting on the nanozyme interface stabilizes the binding conformation of nucleic acid aptamers and template ions, overcomes the interference of environmental factors on nucleic acid aptamers and their non-specific adsorption behavior, and improves the recognition selectivity of nucleic acid aptamer-modified nanozymes.

[0023] 2. The synergistic effect between the imprinted layer and the nucleic acid aptamer on the interface improved the catalytic activity of the nanozyme and enhanced the detection sensitivity. Attached Figure Description

[0024] Figure 1 The images show the UV absorption spectra of nano-gold, Au-T30695, and Pb-IIP.

[0025] Figure 2 Au-T30695, Pb-IIP, Pb-NIP and different concentrations of Pb 2+ Linear relationship graph.

[0026] Figure 3 Pb-IIP with different concentrations of Pb 2+ The combination dynamics curve.

[0027] Figure 4 Au-T30695 and different concentrations of Pb 2+ The combination dynamics curve.

[0028] Figure 5 Pb-NIP with different concentrations of Pb 2+ The combination dynamics curve.

[0029] Figure 6 Au-T30695 and Pb-IIP at different Na concentrations + The ability to resist interference. Detailed Implementation

[0030] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings:

[0031] Example 1

[0032] A method for preparing nucleic acid aptamer-modified nanoenzyme ion-imprinted polymer:

[0033] 1) Synthesis of gold nanoparticles

[0034] Pour a 1% HAuCl4 (v / v) aqueous solution into a three-necked flask equipped with a reflux condenser and heat with stirring until boiling. Then quickly add 10 ml of 38.8 mM trisodium citrate. Reflux and heat for another 5 minutes, until the color changes from pale yellow to wine red. Finally, continuously stir the solution and cool it to room temperature.

[0035] 2) Preparation of aptamer-modified gold nanoparticles

[0036] This embodiment uses 5'-modified SH Pb 2+Nucleic acid aptamer (hereinafter referred to as T30695) was obtained by mixing Au NPs solution with SH-T30695 at a ratio of 1:100 and incubating for 22 hours. Then, 0.1M salt was added in 4 portions (one every half hour) and incubated at room temperature for 48 hours.

[0037] The obtained solution (Au-T30695) was centrifuged for 15 min to remove unbound aptamers, and the precipitate was washed with 5 mM pH 8.0 Tris-acetic acid. The precipitate was dispersed in 5 mM pH 8.0 Tris-acetic acid solution and stored at 4 °C.

[0038] 3) Ion-imprinted polymer (Pb-IIP) prepared based on Au-T30695 nanozyme

[0039] Au-T30695 and Pb 2+ Pre-assembly was performed for 40 min, followed by the addition of 0.06 mg / ml dopamine (dissolved in 10 mM pH 8.5 Tris-hydrochloric acid) and polymerization initiation at room temperature for 90 min. The resulting mixture was then centrifuged at 11,000 rpm and 4 °C for 15 min to separate the precipitate particles. The centrifuged product was then washed with an acidic solution to remove Pb. 2+ The mixture was vortexed to completely disperse the lead ions, then placed on a shaker and shaken for 15 minutes to break the binding between the aptamers and lead ions, thus eluting the template. It was then centrifuged at 10,000 rpm for 10 minutes, and the supernatant was discarded. This process was repeated until Pb was undetectable in the washings using an atomic absorption spectrophotometer. 2+ The existence of Pb means that Pb is obtained. 2+ Imprinted polymer Pb-IIP.

[0040] Then, Pb-IIP was dispersed in 1 mL of ultrapure water and vortexed in a test tube to ensure thorough dispersion and mixing. The solution was then shaken on a shaker for 5 min to wash away any residual eluent. The solution was then centrifuged at 10,000 rpm for 10 min and redispersed in water until the aqueous solution of the imprinted polymer was neutral. Finally, the solution was resuspended in 5 mM Tris-acetic acid solution at pH 8.0.

[0041] The preparation of the non-imprinted polymer Pb-NIP follows the same steps as described above, the only difference being that it does not require contact with Pb. 2+ Pre-assembled.

[0042] Example 2

[0043] Interactions of Au-T30695, Pb-IIP, Pb-NIP with Pb(II): The three particles were subjected to 0.05-100 nmol / L Pb 2+The solutions were reacted separately, and then 100 μM Amplex UltraRed fluorescent substrate and 4 mM H2O2 were added and incubated at 27 °C for 2 h. The fluorescence intensity at 584 nm was recorded under 540 nm excitation, thus establishing the Pb concentration for different concentrations. 2+ The linear relationship between the fluorescence intensity of the solution and the change in fluorescence intensity.

[0044] Among them, K of Pb-IIP and Au-T30695 m and V max The values ​​are shown in the table below:

[0045]

[0046] The adsorption kinetic fitting parameters for Au-T30695, Pb-IIP, and Pb-NIP are shown in the table below:

[0047]

[0048] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a nucleic acid aptamer-modified nanoenzyme ion-imprinted polymer, characterized in that, Includes the following steps: Step 1: Preparation of gold nanoparticles; Step 2: Combine the prepared gold nanoparticles with Pb 2+ Nucleic acid aptamers are co-incubated, sodium salt is added, and incubation is repeated. Excess nucleic acid aptamers are removed by centrifugation to obtain nucleic acid aptamer-modified nanozymes, namely non-imprinted polymers Pb-NIP. Step 3: Combine the aptamer-modified nanozyme obtained in Step 2 with Pb 2+ Pre-assembly, followed by the addition of a functional monomer and initiation of polymerization, and finally centrifugation to obtain precipitated particles; the functional monomer is dopamine. Step 4: Wash the centrifuged product with an acidic solution to remove Pb. 2+ Until no Pb is found in the washing solution. 2+ Upon detection, the nucleic acid aptamer-modified nanozyme ion-imprinted polymer Pb-IIP is obtained.

2. The method for preparing the nucleic acid aptamer-modified nanoenzyme ion-imprinted polymer as described in claim 1, characterized in that, Step 2 must satisfy one or more of the following conditions: Condition 1 is gold nanoparticles and Pb 2+ The molar ratio of nucleic acid aptamers is 1:30 to 1:500; Condition 2 is that the co-incubation time is 20h~24h; Condition 3 is that the concentration of the sodium salt is 0.1M; Condition 4 is that the addition is done every half hour, for a total of four times; Condition 5 is that the incubation time is 40-48 hours.

3. The method for preparing the nucleic acid aptamer-modified nanoenzyme ion-imprinted polymer as described in claim 1, characterized in that, Step 3 described above satisfies one or more of the following conditions: The concentration of the functional monomer described in condition 1 is 0.01~1 mg / mL. -1 ; Condition 2 is that the polymerization time is 30 min to 360 min.

4. A nucleic acid aptamer-modified nanozyme ion-imprinted polymer as described in any one of claims 1 to 3 in Pb 2+ Its application in detection is characterized by, Includes the following steps: Nucleic acid aptamer-modified nanozyme ion-imprinted polymer Pb-IIP with different concentrations of Pb 2+ The solutions were mixed, followed by the addition of Amplex UltraRed fluorescent substrate and H2O2 for co-incubation. Fluorescence intensity at 584 nm was recorded under 540 nm excitation to establish a system for different concentrations of Pb. 2+ The linear relationship between the fluorescence intensity of the solution and the change in fluorescence intensity.

5. The nucleic acid aptamer-modified nanozyme ion-imprinted polymer as described in claim 4 in Pb 2+ Its application in detection is characterized by, The co-incubation temperature is 25~37℃, and the time is 2h.

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